Abstract: :
Purpose:Retinal neurons are known to be extensively coupled,both homologously and heterologously, through gap junction intercellularchannels, but little is known about connexin subtypes expressedby mammalian retinal neurons. In addition, retinal gap junctioncoupling is known to be modulated by both dopamine and NO throughconnexin phosphorylation, indicating that at least some of theretinal connexins are phosphoproteins. In this work, we exploredwhether the connexin phosphoprotein subtype, connexin 40, wasexpressed in mammalian retina.Methods:In immunofluorescentstaining experiments, light-adapted bovine and rat retinas werefixed in paraformaldehyde, and 6-8 micron slices permeabilizedwith methanol were reacted with connexin-specific antibodiesfor 24 hr. Reaction was visualized with rhodamine or fluoresceinsecondary antibodies. Puncta were observed using a 100X oilimmersion lens. In Western blot experiments, bovine retinalmembranes were alkaline-extracted, proteins separated on 12.5%acrylamide gels, transferred to PVDF membranes, and incubatedwith connexin-specific antibodies. Bands were detected usingbiotinylated secondary antibodies, AP-conjugated streptavidinand BCIP/NBT.Results:Immunostaining experiments using connexin40-specific antibodies from two independent sources showed punctatestaining between cells in the outer nuclear layer, as well asbetween cells within a sublayer of the inner nuclear layer.Punctate staining in these two regions was observed in approximately40-50% of retinal sections scanned. In addition, sparse punctatestaining was detected in the GCL/AFL region. No punctate stainingwas observed in the outer or inner segment layers in eitherrat or bovine retinas. Double immunostaining of bovine retinaswith antibodies to Go, which stains bipolar cells, and to connexin40, showed little overlap. Western blot analysis of bovine retinasrevealed an immunopositive doublet at approximately 40 kD thatco-migrated with a doublet present in heart membranes. Theseimmunopositive bands disappeared when antibody was pre-incubatedwith a peptide containing the connexin sequence to which theantibody was made.Conclusion:These results suggest that connexin40 is expressed within the neural layers of bovine and rat retinas.Specific cell types expressing connexin 40 remain to be determined.